Annexin A2 binds to vimentin and contributes to porcine reproductive and respiratory syndrome virus multiplication.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important globally distributed and highly contagious pathogen that has restricted cell tropism in vivo and in vitro. In the present study, we found that annexin A2 (ANXA2) is upregulated expressed in porcine alveolar macrophages infected with PRRSV. Additionally, PRRSV replication was significantly suppressed after reducing ANXA2 expression in Marc-145 cells using siRNA. Bioinformatics analysis indicated that ANXA2 may be relevant to vimentin, a cellular cytoskeleton component that is thought to be involved in the infectivity and replication of PRRSV. Co-immunoprecipitation assays and confocal analysis confirmed that ANXA2 interacts with vimentin, with further experiments indicating that the B domain (109-174 aa) of ANXA2 contributes to this interaction. Importantly, neither ANXA2 nor vimentin alone could bind to PRRSV and only in the presence of ANXA2 could vimentin interact with the N protein of PRRSV. No binding to the GP2, GP3, GP5, nor M proteins of PRRSV was observed. In conclusion, ANXA2 can interact with vimentin and enhance PRRSV growth. This contributes to the regulation of PRRSV replication in infected cells and may have implications for the future antiviral strategies.

Correction to: Annexin A2 binds to vimentin and contributes to porcine reproductive and respiratory syndrome virus multiplication.

In the original publication of this article [1], the author found the brand of vimentin antibody was wrong in Fig. 3. The legend of Fig. 3, 'mouse anti-vimentin mAb (Cell Signaling Technology) at 4 °C overnight' should be 'mouse anti-vimentin mAb (Sigma-Aldrich) at 4 °C overnight'.

Importation and Recombination Are Responsible for the Latest Emergence of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus in China.

In China, a majority of the highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRSV) strains were seeded by the 2006 outbreak. However, the most recently emerged (2013-2014) HP-PRRSV strain has a very different genetic background. It is a NADC30-like PRRSV strain recently introduced from North America that has undergone genetic exchange with the classic HP-PRRSV strains in China. Subsequent isolation and characterization of this variant suggest high pathogenicity, so it merits special attention in control and vaccine strategies.

Influence of acupuncture on neural movement function in rats with middle cerebral artery occlusion--a randomized controlled trial.

OBJECTIVES: To observe recovery in movement function in rats with middle cerebral artery occlusion (MCAO) after acupuncture treatment. METHODS: According to the randomized and controlled principle 1384 rats were divided into the basic control group (including the normal, sham, model control, model without intervention, Nimodipine, and para-Renzhong groups) and the acupuncture group (including the Neiguan (PC 6), Weizhong (BL 40), Sanyinjiao (SP 6), Chize (LU 5), Renzhong (GV 6) and non-acupoint groups). MCAO was modeled by Zea-longa's thread ligation and rats with scores of 1-3, as assessed by Zausinger's six-point method, were used in this study. Moreover, in the acupuncture group each acupoint was set with 12 different parameters by the orthogonal intersection method, resulting in 78 groups with 18 rats per group. The rats were treated by acupuncture once every 12 h for a total of six sessions and neurobehavioral scores were measured after each session. The neurobehavioral scores were compared by one-way ANOVA using the statistical software SPSS 17.0. RESULTS: After acupuncture therapy the mean neurobehavioral scores in MCAO rats increased gradually at each time point with a significant difference among the six scores, but with no significant differences between the fourth (48 h) and the fifth score (60 h), and between the fifth (60 h) and the sixth (72 h) score (P > 0.05). CONCLUSIONS: MCAO rats gradually recovered movement function over multiple acupuncture sessions. After the fouth acupuncture session (48 h), the neurobehavioral scores of rats with cerebral infarction remained stable. Acupuncture treatment had a reliable curative effect on movement function in cerebral infarction rats.

[Operation-effect analysis of 21 patients with ankle fracture and complete rupture of deltoid ligament].

OBJECTIVE: To explore short-term clinical effect of surgical treatment for ankle fracture with complete rupture of deltoid ligament in young and middle-aged patients. METHODS: From January 2016 to March 2019, 21 young and middle-aged patients with ankle joint fracture and complete rupture of the deltoid ligament were treated with internal fixation and repair of the medial deltoid ligament, including 16 males and 5 females, aged from 21 to 52 years old with an average of (38.6±7.3) years old, the time from injury to operation ranged from 1 to 7 days with an average of (3.8±1.5) days. Fracture healing time and complications were observed, changes of medial malleolus clearance before and after operation were compared, American Orthopaedic Foot and Ankle Society (AOFAS) score wasused to evaluate function of ankle joint at 18 months after operation. RESULTS: Twenty-one patients were followed up from 18 to 26 months with an average of (21.7±1.2) months. The incisions were healed at stageⅠ, and fracture healing time ranged from 8 to 12 weeks with an average of (9.5±1.6) weeks. No wound infection, failure of internal fixation, and nerve injury occurred. Medial malleoius space decreased from (5.83±0.32) mm before operation to (2.69±0.25) mm after operation. Postoperative AOFAS score at 18 months was 91.43±4.14, 14 patients got excellent results, 6 good and 1 fair. CONCLUSION: On the basis of stable fixation of fracture, repair of deltoid ligament could help restoring the medial anatomy of ankle joint in young and middle-aged patients, and could achieve good short term clinical effect.

Genotypic and geographical distribution of porcine reproductive and respiratory syndrome viruses in mainland China in 1996-2016.

Porcine reproductive and respiratory syndrome (PRRS) has caused huge economic losses to Chinese swine industry and remains a major threat since it was first reported in 1996. However, investigations of molecular epidemiological and genetic diversity of PRRS viruses (PRRSVs) in China were limited to a small number of representative strains collected in several areas. Moreover, lineage classifications reported by individual researchers were quite different. In the present study, we sequenced ORF5 sequences of 217 PRRSVs from clinical samples, retrieved all the available ORF5 sequences of PRRSVs isolated in China in 1996-2016 (n=2213) from GenBank, and systematically analyzed corresponding epidemiological data. NA-type PRRSVs in China were classified into five lineages: lineage 1, lineage 3, lineage 5, lineage 8, and lineage 9. Most strains in China belonged to lineage 8 (85.6%), with dominant strains being classified as sublineage 8.3 (78.3%). Importantly, the emerging lineage 1 and lineage 3 strains spread rapidly, and their proportions among circulating PRRSVs have significantly increased in recent years. The geographical distribution of different PRRSV lineages in each province was analyzed and possible inter-province transmission routes were outlined for main lineages and sublineages. To our knowledge, this study is the most comprehensive and extensive phylogeographical analysis of PRRSVs in China since PRRS outbreak in 1996. Our dataset can serve as a canonical standard for PRRSV classification and will help to study genetic evolution of PRRSV. The results of the present study may also improve prevention of PRRS in China.

Highly Efficient CRISPR/Cas9-Mediated Homologous Recombination Promotes the Rapid Generation of Bacterial Artificial Chromosomes of Pseudorabies Virus.

Bacterial artificial chromosomes (BACs) are powerful tools for the manipulation of the large genomes of DNA viruses, such as herpesviruses. However, the methods currently used to construct the recombinant viruses, an important intermediate link in the generation of BACs, involve the laborious process of multiple plaque purifications. Moreover, some fastidious viruses may be lost or damaged during these processes, making it impossible to generate BACs from these large-genome DNA viruses. Here, we introduce the CRISPR/Cas9 as a site-specific gene knock-in instrument that promotes the homologs recombination of a linearized transfer vector and the Pseudorabies virus genome through double incisions. The efficiency of recombination is as high as 86%. To our knowledge, this is the highest efficiency ever reported for Pseudorabies virus recombination. We also demonstrate that the positions and distances of the CRISPR/Cas9 single guide RNAs from the homology arms correlate with the efficiency of homologous recombination. Our work show a simple and fast cloning method of BACs with large genome inserted by greatly enhancing the HR efficiencies through CRISPR/Cas9-mediated homology-directed repair mechanism, and this method could be of helpful for manipulating large DNA viruses, and will provide a successful model for insertion of large DNA fragments into other viruses.

Genomic analyses reveal that partial sequence of an earlier pseudorabies virus in China is originated from a Bartha-vaccine-like strain.

Pseudorabies virus (PRV), the causative agent of Aujeszky's disease, has gained increased attention in China in recent years as a result of the outbreak of emergent pseudorabies. Several genomic and partial sequences are available for Chinese emergent and European-American strains of PRV, but limited sequence data exist for the earlier Chinese strains. In this study, we determined the complete genomic sequence of one earlier Chinese strain SC and one emergent strain HLJ8. Compared with other known sequences, we demonstrated that PRV strains from distinct geographical regions displayed divergent evolution. Additionally, we report for the first time, a recombination event between PRV strains, and show that strain SC is a recombinant of an endemic Chinese strain and a Bartha-vaccine-like strain. These results contribute to our understanding of PRV evolution.

Genomic characterization of emergent pseudorabies virus in China reveals marked sequence divergence: Evidence for the existence of two major genotypes.

Recently pseudorabies outbreaks have occurred in many vaccinated farms in China. To identify genetic characteristics of pseudorabies virus (PRV) strains, we obtained the genomic sequences of PRV strains HeN1 and JS, which were compared to 4 PRV genomes and 729 partial gene sequences. PRV strains isolated in China showed marked sequence divergence compared to European and American strains. Phylogenetic analysis revealed that for the first time PRV can be divided into 2 distinct clusters, with Chinese strains being genotype II and PRVs isolated from other countries being genotype I. Restriction fragment length polymorphism analysis confirmed differences between HeN1 and Bartha strains, as did the presence of unique insertion/deletion polymorphisms and microsatellites. This divergence between the two genotypes may have been generated from long-term, independent evolution, which could also explain the low efficacy of the Bartha vaccine in protecting pigs infected with genotype II PRV.

Study on acupuncture parameters impacting on the acupuncture effect using cluster analysis in a rat model with middle cerebral artery occlusion.

OBJECTIVE: To investigate the influence of acupuncture parameters on the acupuncture effect through the cluster analysis in Wistar rat model with middle cerebral artery occlusion (MCAO). METHODS: Duplicated MCAO models by Zea-longa's thread ligation and chose rats with 1-3 scores assessed by Zausinger's six-score method to be grouped. The rats were divided into 6 basic control groups [(including a normal group, a sham group, a model control group, a model group without intervention, a Nimodipine group, a lateral-to-Renzhong (DU6) group] and 6 acupuncture groups [a Neiguan (PC6) group, a Weizhong (BL40) group, a Sanyinjiao (SP6) group, a Chize (LU5) group, a Renzhong (DU6) group and a Feixue (non-acupoint) group]. In the acupuncture groups, for every acupoint or needling site, 9 different parameters [2 factors (frequency and time) and 3 levels (180, 120, and 60 cpm of the frequency and 5, 60, and 180 s of the time)] were set respectively by the orthogonal intersection method, in total 54 groups. The rats were treated by acupuncture with a lifting-thrusting manipulation once every 12 h, in total 6 times. Neurobehavioral scores, cerebral blood flow, infarction rate, microcirculation, light microscopy, etc. were measured. The factor analysis was first applied to get the comprehensive effect scores of the samples in the acupuncture groups and then by which the cluster analysis was made with the statistical software of SPSS17.0. RESULTS: For the Neiguan (PC6) group, the exceptional results of acupuncture comprehensive effect were parameters 7, 8, 9, 10; the valid results were parameters 2, 3, 4, and the invalid were parameters 5, 6. For the Weizhong (BL40) group, the exceptional results were parameters 2, 4; the valid results were parameters 3, 5, 6, 7, and the invalid were parameters 8, 9, 10. For the Chize (LU5) group, the exceptional results were parameters 7, 8; the valid results were parameters 3, 4, 5, 6, 9, 10; and the invalid was parameter 2. For the Sanyinjiao (SP6) group, the exceptional results were parameters 4, 6; the valid results were parameters 2, 3, 5; and the invalid were parameters 7, 8, 9,10. For the Renzhong (DU6) group, the exceptional results were parameters 3, 4, 6, 7, 9, 10; the valid results were parameters 2, 5; and the invalid was parameter 8. For the Non-acupoint group, the exceptional result was parameter 10; the valid results were parameters 2, 3, 4, 7, 9; and the invalid were parameters 5, 6, 8. CONCLUSIONS: For each meridian acupoint, different acupuncture parameters could consequently get a different acupuncture effect; each meridian acupoint had the most suitable or optimal acupuncture parameters; acupuncture parameters might be the main factors impacting on acupuncture effect.

Cluster analysis for acupoint specificity of acupuncture effect based on cerebral infarction rat model.

OBJECTIVES: To study the acupoint specificity through the comprehensive evaluation of the acupuncture effect on rat model with the middle cerebral artery occlusion (MCAO). METHODS: Duplicated MCAO model by Zea-longa's thread ligation was assessed by Zausinger's six-point method, and rats with 1-3 scores were chosen to be grouped. According to the randomized and controlled principle, rats were divided into 6 basic control groups (including normal group, sham group, model control group, model group without intervention, Nimodipine group, lateral-to-Renzhong group) and 6 acupuncture groups [including Neiguan (PC6) group, Weizhong (BL40) group, Chize (LU5) group, Sanyinjiao (SP6) group, Renzhong (DU6) group and non-acupoint group]; in each acupoint or non-acupoint 9 different parameter combinations were respectively set by orthogonal intersection method. There were in total 60 groups, and each group had 12 rats. The rats were treated by acupuncture with the lifting-thrusting manipulation every 12 h, in total 6 times. Indices of neurobehavioral scores, cerebral blood flow, infarction rate, microcirculation, and light microscope, etc. were measured. In order to comprehensively evaluate the acupuncture effect to research the specificity of acupoint, a cluster analysis was made with SPSS17.0 for the comprehensive effect scores of the samples computed according to the comprehensive evaluation models, and then sorting and classification were made for the comprehensive effect scores. RESULTS: In the acupuncture groups every acupoint had different therapeutic effect due to different acupuncture parameter combinations; among the acupuncture groups the orders of curative effect were as followings: Neiguan group was of exceptional result, Weizhong group and Sanyinjiao group were of valid results in upper level, Chize group and Renzhong group were of valid results in lower level, and non-acupoint group was of invalid result; the therapeutic result of acupoint group was superior to non-acupoint group; and the curative effect of acupuncture group was also superior to basic control group. CONCLUSION: The acupoint has the specificity of acupuncture effect, and the acupoint specificity is relative.